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K Orth

Publications and source records attributed to K Orth.

At least 73 records · Page 4Linked to original sources
PubMed1991

The primary structures of four subunits of the human, high-molecular-weight proteinase, macropain (proteasome), are distinct but homologous.

Macropain (proteasome) is a high-molecular-weight proteinase complex composed of at least 13 electrophoretically distinct subunits. Previous work, including peptide mapping and limited amino acid sequencing, suggested that most of the subunits belong to an evolutionarily related group of different gene products (Lee et al. (1990) Biochim. Biophys. Acta. 1037, 178-185). In order to define the extent and pattern of subunit relatedness, and to determine the structural basis for possible similarities and differences in subunit functions, we are deducing the primary structures of macropain subunits by cDNA cloning and DNA sequence analysis. We report here the primary structures of four subunits. The data clearly demonstrate that the proteins represent different, but homologous gene products. Surprisingly, no evidence for homology with any other protein, including proteinases, was obtained. These results suggest that macropain is comprised of a previously unidentified family of evolutionarily related polypeptides. Because biochemical data indicate that macropain contains several different proteinase activities, the current results raise the possibility that the macropain complex is composed of a group of novel proteinases, distinct from those of other structurally identifiable proteinase families.

Amino Acid Sequence↗
PubMed1991

Genetic and molecular characterization of tube, a Drosophila gene maternally required for embryonic dorsoventral polarity.

Loss of maternal function of the tube gene disrupts a signaling pathway required for pattern formation in Drosophila, causing cells throughout the embryo to adopt the fate normally reserved for those at the dorsal surface. Here we demonstrate that tube mutations also have a zygotic effect on pupal morphology and that this phenotype is shared by mutations in Toll and pelle, two genes with apparent intracellular roles in determining dorsoventral polarity. We then describe the isolation of a functionally full-length tube cDNA identified in a phenotypic rescue assay. The tube mRNA is expressed maximally early in embryogenesis and again late in larval development, corresponding to required periods of tube activity as defined by distinct maternal and zygotic loss-of-function phenotypes in tube mutants. Sequence analysis of the cDNA indicates that the tube protein contains five copies of an eight-residue motif and shares no significant sequence similarity with known proteins. These results suggest that tube represents a class of protein active in signal transduction at two stages of development.

Amino Acid Sequence↗
PubMed1991

[Gallbladder carcinoma as an incidental finding].

Between March 1982 and December 1990, 903 patients underwent elective cholecystectomy. In 40 patients cholecystectomy was performed for gallbladder carcinoma. 15 malignomas (1.7%) were found incidentally. Preoperatively no anamnestic or diagnostic tumor signs were found in this group of patients. An en-bloc-resection of the gallbladder with resection of the bordering liver segments was performed when gallbladder carcinoma was diagnosed intraoperatively. When the diagnosis was established by postoperative histology, a relaparotomy with liver resection and lymphnode-dissection was done, except in one case (T-1a stage). Histology showed adenocarcinoma in 11 out of 15 cases. No significant difference in the course of the disease was observed in patients with gallbladder carcinoma of different types. The hospital mortality rate was 0% after curative and palliative surgical treatment of gallbladder carcinoma. Patients with T-1 and T-2 stage have survived without tumor-recurrence up to now. The median survival time after surgery for gallbladder carcinoma in T-3 stage was 17 months, and 8 months in T-4 stage. The morbidity rate after elective cholecystectomy is low and hospital mortality is 0%. According to the short survival times in advanced stages of gallbladder carcinoma, a prompt cholecystectomy in symptomatic gallbladder lithiasis or chronic cholecystitis is advocated.

Adenocarcinoma↗
PubMed1990

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae.

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.

Amino Acid Sequence↗
PubMed1990

Relationships among the subunits of the high molecular weight proteinase, macropain (proteasome).

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologous. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the 'latent' and 'active' forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.

Amino Acid Sequence↗
PubMed1990

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding.

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1990

Comparison of the soluble and membrane-bound forms of the puromycin-sensitive enkephalin-degrading aminopeptidases from rat.

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.

Amino Acid Sequence↗
PubMed1990

[Cholecystectomy--the gold standard of therapy].

Elective CCE is still the treatment of choice for more than 80% of patients with gallstone disease. Surgery is indicated for biliary symptoms along with sonographic or radiographic evidence of gallstones and/or pathology of the gallbladder wall. Non-operative procedures are limited to patients with open cystic duct, preserved contractivity of the gallbladder and non-calcified stones. Mortality of elective cholecystectomy in patients less than 70 years today is 0. Emergency cholecystectomy and old age (greater than 70) are the only mortality risk factors amounting up to 4%. Recurrent surgery is necessary in less than 1% of the patients, conservatively treated postoperative morbidity less than 5%. Cholecystectomy still remains the only method, by which immediate stone release is achieved and long term medication avoided.

Cholecystectomy↗
PubMed1990

[Risk in elective cholecystectomy].

From 3/1982 to 12/1989 773 patient underwent an elective Cholezystectomy at the Surgical University Hospital of Ulm. In 87% of cases the indication was a symptomatic cholelithiasis, the mean age being 55.4 years for females and 56.6 years for males. Intraoperative complications were rare (2.8%) and of a technical nature. Postoperative morbidity was 15.2%. General disturbances occurred in 10.3% and were age-depended. Local postoperative problems (4.9%) did not show this age-dependent distribution. Residual stones were detected in 0.8% of cases; they were removed operatively or endoscopically or crushed by shock waves. 1.3% of patients had to be re-operated. In the age group under 70 years as well as over 70 years lethality was 0%. In 9 cases (1.16%) a gallbladder carcinoma was found incidentally during the operation. We think that elective cholecystectomy is a safe therapeutic procedure in gallbladder stone disease, which also considers the association of chronic cholelithiasis and gallbladder carcinoma.

Adolescent↗
PubMed1989

Molecular cloning of a histidine-rich Ca2+-binding protein of sarcoplasmic reticulum that contains highly conserved repeated elements.

We reported previously the purification of a 165-kDa muscle-specific protein identified by virtue of its ability to bind 125I-labeled low density lipoprotein with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Hoffmann, S. L., Brown, M. S., Lee, E., Pathak, R. K., Anderson, R. G. W., and Goldstein, J. J. (1989) J. Biol. Chem. 264, 8260-8270). The protein is located in the lumen of the sarcoplasmic reticulum, where it has no access to plasma lipoproteins. It binds to 45Ca2+ on nitrocellulose blots and stains metachromatically blue with Stains-all, a cationic dye that stains Ca2+-binding proteins. In the current paper, we have isolated a full-length rabbit cDNA clone for the 165-kDa protein. The deduced amino acid sequence reveals a 852-amino acid protein with the following structural features: 1) an NH2-terminal 27-residue putative signal sequence; 2) a highly repetitive region containing nine nearly identical tandem repeats of 29 residues, each consisting of a histidine-rich sequence HRHRGH, a stretch of 10-11 acidic amino acids, and a sequence containing 2 serines and a threonine in a negatively charged context; 3) a 13-residue stretch of polyglutamic acid; and 4) a COOH-terminal cluster of 14 closely spaced cysteine residues with the repeating pattern of Cys-X-X-Cys suggestive of a heavy metal binding domain. Histidine, aspartic acid, and glutamic acid accounted, respectively, for 13, 12, and 19% of the amino acids. The protein does not share any significant sequence homology with the cell surface low density lipoprotein receptor. Stretches of acidic amino acids are a feature of two other luminal sarcoplasmic reticulum proteins, suggesting that these may be a general feature of luminal sarcoplasmic reticulum proteins. We suggest that the histidine-rich Ca2+-binding protein described in the current study be designated HCP. The role of HCP in Ca2+ homeostasis in the sarcoplasmic reticulum of skeletal and cardiac muscle remains to be determined.

Amino Acid Sequence↗
PubMed1989

[Splenectomy of the giant spleen].

From 1982 to 1987 splenectomy was performed in 45 patients with massive splenomegaly (greater than 1.5 kg). Only three patients could be cured surgically. In 42 patients a serious hematological disorder was the indication for operation, and splenectomy served as palliative treatment of mechanical complaints and to achieve a correction of cytopenias. 56% of the patients sustained postoperative complications, mainly respiratory problems and wound infections. None of the patients died as a result of the operation.

Adolescent↗
PubMed1989

Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure.

Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.

Adenylyl Cyclases↗
PubMed1989

Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum.

The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.

Amino Acid Sequence↗
PubMed1989

Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b.

The P2 porin protein is the most abundant protein in the outer membrane of Haemophilus influenzae type b (Hib). Biochemical and immunochemical techniques were used to characterize the P2 proteins from a number of different Hib strains. P2 proteins from Hib outer membrane vesicles were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for in situ tryptic digestion. Solid-phase tryptic digests of P2 from eight Hib strains were resolved by high-pressure liquid chromatography and shown to be similar if not identical. Radioimmunoprecipitation analysis involving Hib cells (containing intrinsically radiolabeled proteins or lipooligosaccharide) and Western blot (immunoblot) analysis were used to identify two P2-specific murine monoclonal antibodies (MAbs). These MAbs were shown to be reactive with 120 Hib strains tested in a colony blot radioimmunoassay. One of these MAbs bound to a surface-exposed P2 epitope that was antibody accessible on all Hib strains tested; the other MAb was directed against a P2 epitope that either was not exposed on the cell surface or was otherwise inaccessible to antibody.

Amino Acid Sequence↗
PubMed1989

Primary structure of the porin protein of Haemophilus influenzae type b determined by nucleotide sequence analysis.

Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib). The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the Hib P2 gene was significantly different from that observed for a gene encoding a porin of E. coli. DNA hybridization studies indicated that there is a single copy of the P2 gene in the Hib chromosome. The availability of the nucleotide and amino acid sequences for the Hib P2 protein will facilitate investigation of the antigenic characteristics and structure-function relationship of this porin.

Amino Acid Sequence↗
PubMed1988

Multiple genes encode nuclear factor 1-like proteins that bind to the promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

DNA-binding proteins of the nuclear factor 1 (NF1) family recognize sequences containing TGG. Two of these proteins, termed reductase promoter factor (RPF) proteins A and B, bind to the promoter for hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a negatively regulated enzyme in cholesterol biosynthesis. In the current study, we determined the sequences of peptides derived from hamster RPF proteins A and B and used this information to isolate a cDNA, designated pNF1/Red1, that encodes RPF protein B. The peptide sequence of RPF protein A, the other reductase-related protein, suggests that it is the hamster equivalent of NF1/L, which was previously cloned from rat liver. We also isolated a hamster cDNA for an additional member of the NF1 family, designated NF1/X. Thus, the hamster genome contains at least three genes for NF1-like proteins. It is likely to contain a fourth gene, corresponding to NF1/CTF, which was previously cloned from the human. The NH2-terminal sequences of all four NF1-like proteins (NF1/Red1, NF1/L, NF1/X, and NF1/CTF), which are virtually identical, contain the DNA-binding domain that recognizes TGG. Functional diversity may arise from differences in the COOH-terminal sequences. We hypothesize that the COOH-terminal domain interacts with adjacent DNA-binding proteins, thereby stabilizing the binding of a particular NF1-like protein to a particular promoter. This protein-protein interaction confers specificity to a class of proteins whose DNA-recognition sequence is widespread in the genome. Sterols may repress transcription of the reductase gene by disrupting this protein-protein interaction.

Amino Acid Sequence↗
PubMed1986

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

Animals↗